Has anyone experienced isolated patches of bleaching in frozen cryostat sections (20- 30um thick) that have been immunostained for TH (chemicon AB151)? We are staining the substantia nigra, VTA and RRF and often find that although we generally get lovely staining in these areas often there are patches where there are obviously lots of neurons but the staining appears very faint i.e. it looks like the colour has washed out. we use a general immuno protocol with strep-HRP and AEC for our staining. we did not experience this when we first started using TH. we have tried a new batch of TH and have gradually replaced most of our other chemicals so we dont think its casued by a bad chemical. does anyone have any suggestions as to what could be causing this?we do include an incubation with 50% ethanol during immuno could this be the problem?oh and the bleaching is occurring as soon as we stain it so its not the AEC fading over time.